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Posted: February 20, 2008

Risks of nanotechnology remain uncertain

(Nanowerk News) Toxicology experiments on nanomaterials often seem to run the same way: put some nanoparticles, carbon nanotubes, quantum dots, or other kind of nanosized structures in a petri dish, water column, soil sample, or lab test tube of choice. Then expose daphnids, microbes, zebrafish, pig lung cells, human skin cells, or other model organisms to the new and exciting materials. Sit back and see what happens.

The peer-reviewed literature contains thousands of articles documenting results from these kinds of tests, all conducted in an effort to determine the health and safety of nanomaterials. Yet the scientific community has yet to determine which nanomaterials are hazardous to the environment or humans, because of a lack of methodology, metrology, and other basics, including how to actually monitor nanoparticles in air, for example. The diversity of nanomaterials, both existing ones and those to come, also presents a challenge.

Researchers say that the field of ecotoxicology and environmental risk assessment of nanomaterials is still in its infancy after less than a decade of concerted effort. And while snapshots from short-term exposure studies are yielding tantalizing glimpses now, the whole picture provided by long-term data on more subtle effects of nanomaterials is completely missing. New methods and collaborations could bring more definitive information soon. Until then, efforts to understand the hazards of nanomaterials continue in a piecemeal fashion.

Experiments in a bucket

Scientists studying nanomaterials have been "learning on the job," says the U.K. Environment Agency's Richard Owen, who recently published a viewpoint article in ES&T about environmental risk assessment of nanomaterials (2007, 41, 5582–558). They mix organisms and nanoparticles, thrown into distilled water or seawater, under conditions that may or may not be similar to natural settings. Then they ask, "'Did the daphnids die, did the fish die?' . . . Researchers use standard methods in simple systems," Owen says, "taking a reductionist approach using tools that are available to them."

Such tactics may be the only option for now. "People are looking for paradigms, and they're starting with research they can do," says Patricia Holden, an environmental microbiologist at the University of California Santa Barbara. "Similar experiments, slightly different materials, similar results—that's a positive thing in a way," she continues. "We're seeing consensus on some aspects of the research, across labs—and a little bit of consistency means the potential for paradigms."

But nailing down behaviors and mechanisms remains tricky. For example, TiO2 can be innocuous in soils on its own but problematic in water or once a coating is added. "People are still grappling with size versus functionality, size versus surface chemistry, and the characteristics that go with each one of those subdivisions," Holden adds. Once those characteristics can be identified, "then maybe we're getting somewhere."

Even if progress is made in the laboratory, extrapolating lab results to the real world will be difficult. Most lab experiments require incredibly high concentrations of nanoparticles or other nanomaterials to kill an organism. For example, a paper published January 16 in ACS Nano (2008, DOI 10.1021/nn700185t) reported that a lethal dose for rats of single-walled carbon nanohorns, or tiny tubules that agglomerate into beautiful dahlia-shaped spheres, topped 2000 milligrams (mg) per kilogram of the animals' body weight. The rats' lung tissues remained undamaged after a 90 day test, despite some blackening from the nanohorns.

But if that test had been conducted for longer, the researchers conjecture, they may have observed some deleterious effects. And such longer term tests are currently lacking in the literature. "It may be that chronic impacts will be more important," Owen says, "or interactions of nanomaterials that change their properties."

For example, bucket experiments have shown that the LC50—or lethal concentration for half a population—for daphnids in water exposed to nanosized TiO2 is 100 mg per liter, a level highly unlikely to occur in the environment. But sometimes effects occur at a smaller, subacute scale: subtle changes to fish gills exposed to copper nanoparticles (Environ. Sci. Technol. 2007, 41, 8178–8186), inflammation in human lung tissues in contact with nanoparticles (e.g., Environ. Sci. Technol. 2007, 41, 331–336), or potential changes over generations because of damage to embryos' or microbes' DNA when nanoparticles get into the cells (e.g., Nano Lett. 2007, 7, 3592–3597).

Same old, same old

Despite the large number of nanomaterials with varying shapes, properties, and sizes, most nanotoxicity experiments remain focused on the known. Typical subjects are lab-synthesized TiO2, fullerenes (or C60), and carbon nanotubes, as well as nanosized silver, copper, and cadmium—all metals known to be toxic at what might be considered their normal molecular sizes but still unknown actors to some extent at the nanoscale.

But even familiar TiO2 presents problems because of its variability, notes Greg Lowry of Carnegie Mellon University. TiO2 has three phases—rutile, anatase, and brookite—each with distinct molecular arrangements that lead to different crystalline characteristics. Each phase can show up in unpredictable proportions at nanoscales. Such unknowns can make reference materials particularly hard to develop for agencies like the U.S. National Institute of Standards and Technology. That variability makes comparisons between studies difficult and lab protocols difficult to universalize.

"We're in a rut right now," says Lowry. While ACS Nano and other journals publish a new nanomaterial with a new shape or coating seemingly every day, scientists "don't have the proper methodologies to fully understand mechanisms of C60 toxicity" and other seemingly "boring" nanomaterials, he says.

New tools

Thilo Hofmann of the University of Vienna suggests that the confluence of geochemists, toxicologists, biologists, and other specialists investigating nanomaterials will present opportunities to adapt techniques across disciplines. For example, cryotechniques—in which samples are frozen and then thin-sectioned for examination with transmission or scanning electron microscopes—may be typical in biology but are seldom used by geochemists and colloids researchers looking at nanomaterials in natural settings. "All systems that need sample preparation change the status of agglomeration" of nanomaterials, Hofmann says. "We want them as they are in nature."

Several researchers are developing new analytical tools for nanotoxicity research. Jennifer Field of Oregon State University and colleagues recently developed a liquid chromatography electrospray ionization mass spectrometry method for tracking a single fullerene in a zebrafish embryo (Anal. Chem. 2007, 79, 9091–9097). And Lee Ferguson of the University of South Carolina comments that he and his colleagues are taking advantage of "some very unique spectral properties" of carbon nanotubes "not shared by any other form of carbon."

Trying "to detect carbon on a carbon background," whether in sediments or inside an organism, is "like trying to find a piece of wheat in a haystack," Ferguson says. He sees "movement but very little progress" in developing methods to examine nanomaterial behaviors in air, water, and soil. Each medium leads to different exposure pathways that must then be elucidated.

Missing mechanisms

Without new tools and standardized methods, scientists might have a hard time determining mechanisms of action and possible exposure pathways. For now, researchers have focused on the very traits that make nanomaterials attractive for applications in industry and medicine—their ability to enter cells and carry other materials as well as a slew of other behaviors that make nanomaterials potentially damaging for humans and the environment.

For example, the massive surface-to-volume ratio of nanoparticles and nanotubes provides nucleation sites for proteins. That could lead to human diseases such as Alzheimer's, according to research published last year in Proceedings of the National Academy of Sciences U.S.A. (2007, DOI 10.1073_pnas.0701250104) but could also have promise for self-assembly and medical treatments. (This work was conducted under unrealistic conditions: in buffer with low pH, which increased fibrillation, and not in an actual cellular system.)

André Nel, chief of the division of nanomedicine at the University of California Los Angeles, and colleagues have investigated how nanoparticles get into cells (Science 2006, 311, 622–627; ACS Nano 2007, 2, 85–96). They found that the same kind of nanoparticle gets taken up by various cell types through diverse pathways. In some cases those interactions can lead to cell death, through very different mechanisms.

"At this stage, we are fortunate in not having any clinical toxicity in humans that we can call up from any engineered nanomaterial," Nel says, "but if we look at what chemical and air pollution can do, then we have to entertain [the possibility] that nanomaterials may show up with [human-health effects] in the future." Environmental effects will come first, he emphasizes, even if they are not observed first.

Surveying the nanolandscape

Emerging databases, modeling efforts, and surveys are beginning to look more closely at the entire life cycle of nanomaterials and how they might get into the environment.

Holden and her colleagues recently surveyed nanomaterials manufacturers in the U.S., Europe, China, and elsewhere to find out whether they were performing their own toxicology testing (Environ. Sci. Technol. 2008, DOI es702158q). "A lot said 'no'," Holden says. Of more than 80 companies that responded to the questionnaire, most reported that they did not do any nanospecific monitoring for workplace health, she adds.

Voluntary reporting and commercial claims for some products underscore the uncertainty about what's out there and make calculating the risks even more difficult. Wendelin Stark of the Swiss Federal Institute of Technology Zurich recalls testing the bathroom cleanser Magic Nano after it caused nearly 100 people in Germany to be hospitalized for respiratory ailments in 2006. His team found the effects were brought on not by the nanoparticles claimed to be in the product (there weren't that many) but by what Stark calls a "classical chemical ingredient" in the product. "No one knew it was the spray," he recalls, which didn't have much "nano" about it except its name.

Further complicating matters, because nanomaterials are often considered proprietary, researchers don't know what's in the materials they might purchase. But they must find a way to test nanomaterials on the market, despite the variability in size, impurities, and other characteristics. The alternative is to create uniform study samples in the lab, which will not give researchers a realistic view of what could end up in the environment.

Researchers can guess where the hot spots will be—downstream from wastewater treatment plants or near industrial spills from manufacturing sites. But such hot spots have yet to be reported. "There hasn't been a lot of work in the field," Owen adds, and no reports have reached the literature yet. (Researchers at the U.S. Geological Survey and elsewhere have examined waste streams and sites downstream from sewage releases; their peer-reviewed reports may appear in the next few months.)

Testing only the simplest nanomaterials merely scratches the surface, considering the plenitude of materials out there now or on their way to market. Different coatings or "functionalizations" are often applied to nanoparticles and nanotubes; this makes the total number of possible combinations seemingly infinite.

Lowry says that although "it looks like coatings make [iron oxide nanomaterials] less toxic for several specific toxicity endpoints," coated particles "appear to get into the nucleus of the cells, and uncoated don't." Quantum dots, for example, are often coated to help them target certain organs for drug delivery. "Once these things get into organs—and people have shown that they [do]—are they going to have long-term effects? I think they are," Lowry asserts.

To make things even more complicated, the toxicity of naked nanoparticles can vary from one location to the next. For example, nanosized TiO2 kills algae by creating radicals when it is exposed to light, but it tends to agglomerate or end up bound to soils and buried in aquifers, hidden in the dark. The behavior of nanomaterials "may change from environment to environment," says Frank van der Kammer, a geochemist at the University of Vienna. If a particle actually moves into a new setting, its functional activity "may be revived," van der Kammer warns, and such "functional persistence" must be considered in future risk assessments.

"The holy grail in this area has been the idea that we could define the risk based on the functionality of the material," says Marc Wiesner of Duke University. "What we're seeing is that it's much more complex than that: interactions with other materials are going to modify and dominate" nanomaterial behaviors.

Lowry says that green chemistry may be the solution to reining in nano risks and hazards. "We could do the right thing by controlling the coverings we use," he says—but even the toxic mechanisms of coatings remain unexplored.

Fast forward

The ongoing deluge of papers, published in a wide array of journals, and continuing discussions make it difficult to keep track of all the data necessary to determine the hazards of nanotechnology. Despite new reviews and continuing databases, not even concerted efforts like the one by the International Council on Nanotechnology at Rice University can keep up, and no one government agency seems to be gathering information for one-stop shopping. In January, the U.S. EPA called for companies to submit data to the Nanoscale Materials Stewardship Program—but such voluntary efforts already under way in the U.K. have yielded relatively little data, and manufacturers continue to pump out ever more nanomaterials.

Slowing down the pace of development and adoption of nanotechnology is not possible, but even so, researchers and policy makers emphasize that they now have a rare opportunity to think about nanotechnology's impacts before its wholesale adoption. "If we can now put the predictive process in place, we should eventually be able to do a prediction survey comparable to the growth of the industry," Nel says. "It will take a couple of years, but it's going to be with us for hundreds of years."

A lack of funding continues to hamper efforts, Nel emphasizes. "It's a pitiful amount that is spent at the moment," he says. "While the proposed amount for human and environmental health and safety should be close to 10% of the entire nano budget, current public spending is less than 1% of this amount."

More investment is imminent from the U.S. National Science Foundation, which is close to completing its first round of evaluations in a competition for a new nanotechnology center. Funded at $5 million a year for 5 years, researchers there would focus solely on environmental and human-health hazards of nanomaterials. In addition to the center, scientists (seconded by several special interest groups and industry) say that they would like to see a directed federal map for future inquiry, like one of the first laid out in Nature (2006, 444, 267–269) by Andrew Maynard of the Woodrow Wilson Center and some of the major researchers in the field.

Despite ongoing activity, scientists conjecture that it could take up to a decade of more research before anyone is ready to judge which nanomaterials are likely to be hazardous. In the end, "the truth will lie somewhere in the middle," says Pedro Alvarez, a chemist at Rice University. "There will always be some kind of collateral damage. . . . Most likely, the benefits of nanotechnology will outweigh its negative impacts, but that's no excuse for us not to understand the negative impacts."